Figure 6.

NADH plus rotenone increases oxidative damage to complex I subunits. Mitochondria from a CTL frontal cortex samples were incubated for 75 min with constituents added as indicated, washed, and then complex I was immunocaptured and analyzed for protein carbonyls as described in Materials and Methods. A, With [NADH] = 125 μm, rotenone (10 μm) appears to increase DNPH reactivity in bands at 18, 21, 26, 37, 50, and 94 kDa. Hydrogen peroxide only increased DNPH reactivity in the ∼20 kDa band. B, In two other CTL brain mitochondrial fractions incubated with a 10-fold higher [NADH] of 1.25 mm (lanes 1 and 3), the increased DNPH density of the ∼50 kDa band from immunocaptured complex I is not further increased with rotenone (10 μm, lanes 2 and 4), but that of the ∼26 kDa band is clearly increased by rotenone. Lanes 1 and 3 are derived from separate CTL brain mitochondrial preparations incubated with 1.25 mm NADH. Lanes 2 and 4 are derived from the CTL mitochondrial preparations used to generate lanes 1 and 3, but after addition of 10 μm rotenone.